Supercoiling of DNA1. Topology

A. Right handed supercoiling = negative supercoiling (underwinding)B. Left handed supercoiling = positive supercoilingC. Relaxed state is with no bendsD. DNA must be constrained: plasmid DNA or by proteinsE. Unraveling the DNA at one position changes the superhelicity -F. Topology only defined for continuous deformation - no strand breakage

Supercoiling of DNA1. Topology

A. Right handed supercoiling = negative supercoiling (underwinding)B. Left handed supercoiling = positive supercoilingC. Relaxed state is with no bends D. DNA must be constrained: plasmid DNA or by proteinsE. Unraveling the DNA at one position changes the superhelicity -F. Topology only defined for continuous deformation - no strand breakage

Supercoiling of DNA

2. Numerical expression for degree of supercoilingA. Equation Lk=Tw+WrB. L:linking number, # of times that one DNA strand winds about the others strands, is always an integer C. T: twist,# of revolutions about the duplex helix D. W: writhe, # of turns of the duplex axis about the superhelical axis by definition the measure of the degree of supercoilingE. specific linking difference or superhelical density=Lk/Lk0

Supercoiling of DNA

2. Numerical expression for degree of supercoilingA. Equation Lk=Tw+WrB. L:linking number, # of times that one DNA strand winds about the others strands, is always an integer C. T: twist,# of revolutions about the duplex helix D. W: writhe, # of turns of the duplex axis about the superhelical axis by definition the measure of the degree of supercoilingE. specific linking difference or superhelical density=Lk/Lk0

Supercoiling of DNA

2. Numerical expression for degree of supercoilingA. Equation Lk=Tw+WrB. L:linking number, # of times that one DNA strand winds about the others strands, is always an integer C. T: twist,# of revolutions about the duplex helix D. W: writhe, # of turns of the duplex axis about the superhelical axis by definition the measure of the degree of supercoilingE. specific linking difference or superhelical density=Lk/Lk0

Supercoiling of DNA1. Topology

A. Right handed supercoiling = negative supercoiling (underwinding)B. Left handed supercoiling = positive supercoilingC. Relaxed state is with no bends D. DNA must be constrained: plasmid DNA or by proteinsE. Unraveling the DNA at one position changes the superhelicity -F. Topology only defined for continuous deformation - no strand breakage

Supercoiling of DNA

3. DNA compaction requires special form of supercoiling

A. Interwound: supercoiling of DNA in solution

B. Toroidal- tight left handed turns, packing of DNA both forms are interconvertible

Supercoiling of DNA

4. Methods for measuring supercoiling - based on how compact the DNA is

A. Gel electrophoresisi. 1 dimensional ii. 2 dimensional

B. Density sedimentation

Supercoiling of DNA

4. Topoisomerases are required to relieve torsional strainA. Topoisomerases I :

breaks only one strand B. Topoisomerase II :

breaks both strands

Supercoiling of DNA

4. Topoisomerases are required to relieve torsional strainA. Topoisomerases I - breaks only one strand

i. monomeric proteinii. after nicking DNA the 5'-PO4 is covalently linked

to enzyme (prokaryotes) or the 3' end is linked to the enzyme (eukaryotes)

iii. evidence is the formation of catenatesiv. E. coli Topo I relaxes negatively supercoiled DNAv. introduces a change of increments of 1 in writhe

Supercoiling of DNA

4. Topoisomerases are required to relieve torsional strainB. Topoisomerase II - breaks both strands

i. supercoils DNA at the expense of ATP hydrolysis

ii. two subunits: (alpha)2 and (beta)2iii. becomes covalently linked to the alpha

subunitiv. relaxes both negative and positively

supercoiled DNAv. introduces a change in increments of 2

in writhe.